Project Summary Ras is a protein that activates the Raf/MEK/ERK signal transduction pathway in the cell, which plays a central role in the control of cell growth and differentiation. In about 30% of human cancers Ras is defective and the signal is kept on, leading to uncontrolled cell proliferation. The signal is normally turned off by the GTPase activity of Ras itself, which hydrolyzes its bound GTP to GDP. While the biochemical and biophysical aspects of the hydrolysis reaction has been well-studied in wild type and mutant free Ras, it is now clear that its protein binding partner Raf has a strong damping effect on the hydrolysis reaction of the common oncogenic mutant RasQ61L, but not on the wild type. This suggests that it is in the context of the interaction with Raf that the oncogenic Q61 mutants of Ras exert their phenotype. This project aims to investigate whether this is true in vivo using NIH-3T3 fibroblasts, to quantify the effect of Raf on the GTPase reaction of the oncogenic mutants of Ras, and to establish the mechanism through which this effect occurs by using a combination of biochemical and biophysical approaches. The goals will be accomplished in four specific aims. The first will be to determine whether the transforming mutants affect the Ras/Raf and PI3K signal transduction pathways differentially. The second will be to obtain the rates of hydrolysis of Ras and several oncogenic mutants in the presence of Raf and compare them to the rates obtained for the free proteins. The third aim focuses on X-ray crystallography and neutron diffraction to study the intrinsic hydrolysis mechanism in Ras and oncogenic mutants. The fourth aim will elucidate the energetic features of the catalyzed reaction using a combination of quantum- mechanical and molecular dynamics simulations of the Ras/Raf complex. By quantifying the effect of Raf on the hydrolysis reaction that controls one of the major cell proliferation pathways in the cell and elucidating the mechanism through which this occurs, this project will significantly advance knowledge of the fundamental mechanisms that lead to the development of human cancers due to mutations in Ras.